Platform processes are valuable tools in process development. Applying them to complex fusion molecules made up of different structures, however, can lead to unexpected results. EA2 is a unique engineered molecule, and it did not fit well into a platform process. It does, however, have three potential affinity and pseudo-affinity sites, each target site having a platform purification unit operation. One site is the antibody Fc region for Protein A binding, but the product was too sensitive to low pHs for typical Protein A elution conditions, so a high pH elution process was developed instead. The other two pseudo-affinity sites proved to be unusable, as did ion exchange chromatography. The final process that was developed added hydrophobic interaction and mixed mode (HIC and AEX) chromatography polishing, with a solvent and detergent (S/D) treatment instead of the typical low pH virus inactivation. This product points out the need for innovation and flexibility in cases where platforms do not work.

Platform processes are powerful and useful tools in process development. Applying them to complex fusion molecules made up of different structures, however, can lead to unexpected results. The process described below is a good example.

The product, designated EA2, is a unique engineered protein and it does not fit into an overall process platform. However, it has three well-known affinity and pseudo-affinity targets, each with well-known binding and elution conditions. Purifications for these targets are unit operations that are used in platform processes. Thus, the development of a purification process was anticipated to be reasonably rapid and simple.

Capture Step

The three targets are the Fc antibody domain target for Protein A, a target for binding to hydroxyapatite, and an enzyme whose active site binds to a dye ligand. Protein A was selected for the initial capture step because it is widely used in bioprocessing.

Table 1. Small-scale low pH Protein A recoveries

The process started with concentration and diafiltration because of its fairly low harvest titer. The concentrated crude bound readily to Protein A, and total recovery of A280 was good. However, recovery of activity was variable, and usually fairly low (Table 1).

Because low pH sensitivity is a common cause of loss of yield off Protein A, a pH stability study was performed (Figure 1a). Based on these results, a pH minimum of 5 was set to provide a safety margin. Even for relatively short exposures, pH levels of 4 and below are to be avoided. This ruled out using low pH for virus inactivation, and makes using low pH elution from Protein A a nonrobust option, even with a short column residence time and immediate neutralization.

It is known that eluate pH will vary between Protein A resins.1,2 Thus, a number of Protein A resins were screened (Table 1). However, in no case was the eluate pH high enough for practical use.